Mismatch repair is a major contributor to genome stability, and defects in the mammalian pathway are associated with a strong predisposition to tumor development. Despite the importance of this system in mutation avoidance, our understanding of its molecular nature is limited. The goals of this project are to establish the conformations and structures of multi-protein and multi-protein DNA complexes that are the key intermediates in triggering the MutS- and MutL-dependent responses to mismatched base pairs and certain types of DNA damage. Our goals are five-fold: (1) Overexpression systems will be developed for production of molecular complexes and truncated polypeptides for structural analysis in collaboration with the Expression-Molecular Biology (EMB) Core. (2) The conformations and dynamics of multi-protein and multi-protein DNA assemblies involved in the initiation step of mismatch repair will be addressed by single particle electron microscopy and small angle X-ray scattering. The latter studies will exploit the high temporal resolution of the Structural Cell Biology (SCB) Synchrotron Beamline and the SCB Core. (3) The structural basis for the recognition of base-base mispairs, insertion/deletion mispairs, and damaged DNA substrates by eukaryotic MutS alpha and MutL alpha will be addressed by X-ray crystallography. (4) Since the initiation of mismatch repair depends on assembly of multi-protein-DNA complexes (MutS MutL DNA in the bacterial system and MutS alpha MutL alpha PCNA DNA in the eukaryotic reaction) these multi-protein and multi-protein-DNA assemblies will be examined using X-ray crystallography. (5) The structural studies above will reveal residues at protein-protein interfaces as well as those that may be involved in conformational transitions. The significance of these residues will be subjected to biological validation by analysis of the phenotypic consequences of genetic alteration of these residues and by examination of selected mutant proteins at the biochemical level. Supporting genetic and biochemical studies will be pursued under funding already available to the Kolodner and Modrich laboratories and will be leveraged to provide powerful Program interactions in a sustainable structural biology cycle where structurally identified interfaces are tested in vivo by mutational analyses. Furthermore, the expected results will contribute directly to Program interactions with Project 1 (on PCNA as a molecular adaptor), Project 2 (on transcription coupled repair) and Project 4 (on homologous recombination) plus comparative ATP-driven conformational switching with Project 1 (ligase), Project 3 (Rad51), and Project 3 (Rad50).